Introduction
In this study, we evaluated the effect of bone-borne accelerated expansion protocols on sutural separation and sutural bone modeling using a microcomputed tomography system. We also determined the optimum instant sutural expansion possible without disruption of bone modeling.
Methods
Sixteen New Zealand white rabbits, 20 to 24 weeks old, were randomly divided into 4 experimental groups. Modified hyrax expanders were placed across their interfrontal sutures and secured with miniscrew implants located bilaterally in the frontal bone. The hyrax appliances were activated as follows: group 1 (control), 0.5-mm per day expansion for 12 days; group 2, 1-mm instant expansion followed by 0.5 mm per day for 10 days; group 3, 2.5-mm instant expansion followed by 0.5 mm per day for 7 days, and group 4, 4-mm instant expansion followed by 0.5 mm per day for 4 days. After 6 weeks of retention, sutural separation and sutural bone modeling were assessed by microcomputed tomography and quantified. Statistical analysis was performed using Kruskal Wallis and Mann-Whitney U tests and the Spearman rho correlation ( P <0.05).
Results
Median amounts of sutural separation ranged from 2.84 to 4.41 mm for groups 1 and 4, respectively. Median bone volume fraction ranged from 59.96% to 69.15% for groups 4 and 3, respectively. A significant correlation (r = 0.970; P <0.01) was observed between the amounts of instant expansion and sutural separation.
Conclusions
Pending histologic verifications, our findings suggest that the protocol involving 2.5 mm of instant expansion followed by 0.5 mm per day for 7 days is optimal for accelerated sutural expansion. When 4 mm of instant expansion was used, the sutural bone volume fraction was decreased.
Highlights
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Bone-borne accelerated expansion and sutural separation were studied in rabbits.
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Sutural separation was correlated with the amount of instant expansion.
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Greater instant sutural expansion enhanced bone formation.
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The protocol of 2.5 mm of instant expansion resulted in the most sutural bone modeling.
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The protocol of 4 mm of instant expansion resulted in the least sutural bone modeling.
Approximately 8% to 18% of patients in the mixed dentition have transverse maxillary constrictions and are frequently managed with tooth-borne rapid maxillary expansion (RME) appliances. Although tooth-borne RME appliances vary in their designs and rates of expansion, their fundamental mechanisms remain the same. Rapid transverse forces are exerted on the maxillary teeth resulting in interruption and separation of the midpalatal sutures. The latter leads to extensive cellular activity in the sutures and encourages bone remodeling. As tooth-borne RME appliances transmit expansion forces through the teeth, dental and alveolar bone bending also occurs during correction of the skeletal disharmonies. These movements take up the major fraction of total appliance activation, reducing the amount of true skeletal expansion.
Recently, mini-implant assisted bone-borne RME appliances that apply lateral forces directly to the midpalatal sutures were introduced. These appliances produce greater orthopedic effects and fewer dentoalveolar side effects compared with tooth-borne ones. Furthermore, they are well tolerated and easier to use than traditional tooth-borne expanders. The efficacy of bone-borne RME is affected by several factors including the rate of distraction or activation. Clinical activation protocols for tooth-borne expanders may not apply to bone-borne ones. Since there is currently no consensus on conventions for bone-borne expanders, expansion protocols for these appliances warrant investigation. Koudstaal et al stated that when distraction is performed too fast for tooth-borne expanders, collagen fibers lose contact, and no ingrowth of new bone occurs, resulting in nonunion or malunion of the separated sutures. Conversely, if the distraction is too slow, premature bone consolidation can occur, and the required expansion cannot be achieved. The quality and quantity of bone formation therefore depend, partially, on the rate of sutural expansion. Although a higher expansion rate has been associated with greater sutural separation, the exact nature of this association and the maximum instant expansion possible without disruption of suture bone modeling remains uncertain.
Several approaches to examining sutural bone modeling have been taken. Microcomputed tomography (micro-CT) is a nondestructive analytical method that does not require arduous specimen preparations and specific skills in microscopy. With advances in micro-CT technology, higher resolution images with reduced metal artifacts can now be achieved. They are particularly useful for assessing the properties of spongy or trabecular bone, including bone density, trabecular bone thickness, and connectivity. We evaluated the effect of bone-borne accelerated expansion protocols on sutural separation and sutural bone modeling using micro-CT. We also determined the optimum instant sutural expansion possible without disruption of bone modeling. The null hypotheses were as follows: there is (1) no difference in sutural separation and bone modeling between dissimilar expansion protocols, and (2) no optimum instant sutural expansion beyond which sutural bone modeling will be disrupted.
Material and methods
The interfrontal suture of rabbits had been proposed as an animal model for palatal expansion and was selected for our study. These sutures were found to act analogously to the midpalatal sutures. In addition, the haversian systems of rabbits are similar to those of humans, allowing for some extrapolation of results to clinical applications. Furthermore, rabbits are adequately small for use with micro-CT, permitting whole-body insertion into the micro-CT chamber for repeated measurements. The frontal areas were also sufficiently wide for the fixation of the palatal expander and 4 miniscrew implants. Adult rabbits (age, 5-6 months; weight, 3-4 kg) were chosen to reduce confounding factors from continuous growth or bone remodeling. Male rabbits were selected to reduce the effects of hormonal variations and to capitalize on their wider frontal bone.
The study was approved by the Faculty of Medicine Institutional Animal Care and Use Committee, University of Malaya (2015-16/006/DENTAL/R/ASH), and all animal work was performed according to the standards specified by the institutional animal care and use committee of the University of Malaya, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Sixteen New Zealand white rabbits, 20 to 24 weeks old, were selected for the study. Sample size calculation was based on the resource equation method (E). “E” can be measured by the following formula: E = total number of animals − total number of groups. According to this method, a sample size with E between 10 and 20 can be considered as adequate (E was 12 in this study). The adult male rabbits (approximately 3.0-4.0 kg) were procured from a licensed farm by the animal experimental unit of the. University of Malaya. Upon their arrival, the rabbits were examined, weighed, and observed twice daily for 2 weeks until their acclimatization. Environmental conditions were maintained as follows: pathogen-free environment with a temperature of 22°C ± 3°C; humidity of 55% ± 10%; and a 12:12 hour light:dark cycle with lights on at 0700 and off at 1900. The animals were housed singly in cages (Techniplast, Buguggiate, Italy) with paper liners. They received maintenance food (2023 diet; Altromin International, Martinsried, Germany) and reverse-osmosis treated water. They were cared for according to the guidelines of the Animal Research Reporting of In Vivo Experiments. Preoperative intramuscular ketamine, 30 mg per kilogram (Troy Laboratories, Smithfield, Australia), and 3 mg per kilogram of xylozine (Troy Laboratories) were administered. The distraction sites were then anesthetized using marcaine (2 mg/kg; Abbott Laboratories, Chicago, Ill) with 1:200,000 epinephrine. General anesthesia was subsequently accomplished with 1% to 3% isoflurane in a 2:1 oxygen/nitrous oxide mixture administered through special facemasks.
Modified expanders were fabricated by laser welding of 4 stainless steel U loops (0.9 mm) to hyrax expanders (Leone, Firenze, Italy). The mucosa and periosteum at the distraction sites between the anterior and posterior limits of the orbital rims were reflected to expose the interfrontal sutures and frontal bones. The modified expanders were positioned across the interfrontal sutures and secured with 4 miniscrew implants (Dentos, Daegu, Korea), 5.0 mm long and 1.7 mm in diameter, using a manual driver ( Fig 1 ). The surgical flaps were subsequently closed, and the rabbits were given intramuscular kombitrim (1 ml/10 kg) (sulfamethoxazole 200 mg and trimethoprim 40 mg) (Kela Laboratoria, Hoogstraten, Belgium) postoperatively to prevent infection, and meloxicam (0.2 mg/kg; Poly Car Labs, Gujarat, India) to minimize discomfort.
The rabbits with their bone-borne expanders were randomly divided into 4 groups, and the hyrax appliances were activated as follows: group 1 (control), 0.5 mm per day expansion for 12 days ; group 2, 1 mm instant expansion followed by 0.5 mm per day for 10 days ; group 3, 2.5 mm instant expansion followed by 0.5 mm per day for 7 days; and group 4, 4 mm instant expansion followed by 0.5 mm per day for 4 days. Thus, a total expansion of the hyrax appliances of 6 mm was applied in all groups. After active expansion, the screws on the hyrax appliances were fixed with light-cured acrylic and left passive for consolidation. After 6 weeks of retention, sutural separation and bone modeling were assessed by micro-CT.
Before micro-CT imaging, the rabbits were again anesthetized with ketamine and xylazine. Posttreatment scans of the distraction sites were obtained using high resolution in-vivo x-ray micro-CT imaging (XtremeCT; Scanco Medical, Bassersdorf, Switzerland). Serial tomographic images were acquired transverse to the interfrontal sutures at 60 kV(p) and 900 μA. One thousand projections were acquired per rotation with an integration time of 300 ms and a voxel size of 41 μm.
An aggregate of 160 slices located between the anterior and posterior miniscrew implants was selected. A region of interest was selected 3 mm from the midline (left and right) and applied to all samples ( Fig 2 , A ). Three-dimensional (3D) images of the region of interest were automatically reconstructed from these 160 slices ( Fig 2 , C ) using the Scanco software (micro-CT evaluation program, version 6.5.3). A Gaussian filter with a sigma value of 0.9 and support value of 1 was used to reduce image noise. The threshold value was set between 130 (lower value) and 1000 (upper value) units to discriminate between the less-dense newly formed bone and the denser old bone based on a previous similar study and data from our pilot study. Bone-volume fraction (BV/TV) at the suture was then measured using Scanco software.
After segmentation of bone tissues using the above threshold values, a 3D color map of the suture tissue separation was generated. Separation maps of the suture tissue indicate maximum separation values with blue to red colors specifying increasing degrees of tissue separation ( Fig 2 , B ).
To calculate the sutural space volume, micro-CT images were exported into medical imaging software (Mimics, version 17.0; Materialise, Leuven, Belgium). For consistency, the same threshold values (224-1249) were used for all specimens to separate soft tissue from bone tissue. As a result, the soft tissue was highlighted in yellow using the so-called masks. The mask was cropped to select the region of interest and used for all specimens. Region growing (computer-assisted tissue separation) and manual tissue deletion (using the multiple slice editing tool) were used to isolate the region of interest. The software then calculated the sutural soft tissue volume (sutural space volume) by means of voxel addition and reconstructed a 3D image ( Fig 3 ).
The amount of sutural separation was established with the RadiAnt DICOM viewer (version 3.4.1; Medixant, Poznan, Poland). The most anterior and posterior slices for the region of interest were first ascertained to locate the bony outline of the sutures. Sutural separation was then calculated with the distance measurement tool ( Fig 4 ) at the point equidistant between the outer surface and the inner surface of the frontal bone and using an average of 2 separate readings. Intraexaminer reliability for mapping the region of interest sutural space volume and measuring suture separation was assessed by repeating the procedures blindly after 2 weeks. Reliability was evaluated using the Cronbach alpha test.
Statistical analysis
All data were analyzed with the Statistical Package for Social Sciences software (verion 20.0; IBM, Armonk, NY). Normality testing was done using the Shapiro-Wilk test. Because the data were not normally distributed, nonparametric Kruskal Wallis and Mann-Whitney U tests ( P <0.05) were used to determine significant differences in sutural separation, BV/TV, sutural space volume, and sutural tissue separation between treatment groups. Spearman rho correlations ( P <0.05) were also performed to establish the associations between the variables.
Results
During the experiment, no peculiarity in weight gained (range, 0.72-0.97 kg) was observed in the rabbits in the 4 groups. There were also no signs of distraction site infection or animal discomfort from the customized distractors. The overall success rate of the miniscrew implants was 98.44% (63 of 64). Only 1 miniscrew implant was displaced upon direct hyrax activation, caused mainly by operator issues. The dislodged miniscrew implant was promptly replaced with a new one buccal to the original site. Reliability testing showed no significant difference in measurements between the 2 assessment periods (α = 0.946).
Median values were 2.99, 3.12, 3.77, and 4.55 mm for anterior sutural separation, and 2.71, 2.78, 3.61, and 4.27 mm for posterior sutural separation in groups 1 to 4, respectively ( Table ). Median values for average sutural separations were 2.84, 2.92, 3.69, and 4.41 mm for groups 1 to 4, respectively ( Table ). Paired comparisons showed statistically significant differences in sutural separation between all groups. All experimental groups had significantly higher sutural separations than did the control group (group 1). Sutural separations for group 2 (2.82%), group 3 (29.93%), and group 4 (55.28%) were higher than those for group 1. Sutural separation for group 4 was significantly higher than for groups 2 and 3, whereas group 3 had a significantly higher separation than did group 2 ( Fig 5 ). The Spearman correlation test showed a strong, positive, and significant correlation ( r = 0.970; P <0.01) between sutural separation and instant expansion.
